Molecular cloning and characterization of cellulase genes of Ruminococcus flavefaciens strain 186 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North, New Zealand

Abstract

A genomic library of Ruminococcus flavefaciens 186 was constructed using the lambda vector λNM1149. It constituted 2.1 x 104 recombinant clones which was large enough to represent the entire genome of this bacterium. From this library, 26 CMC+ clones were identified after screening about 2500 recombinant clones. These CMC+ clones were divided into four groups according to their insertion fragment size. Partial restriction maps of these clones have been achieved. Eight representative clones from these four groups showed different mapping patterns. One of the three 9 kb insert clones (λCM903) was selected for further study based on its ability to express all three types of cellulase activities. The locations of endoglucanase and exoglucanase genes in λCM903 were determined. Two separate fragments from λCM903 were subcloned and named λCMEH1 (CMC+) and λCMEH2 (MUC+). Cross hybridization experiment using celA gene of C. thermocellum and the 26 recombinant clones showed no significant homology. However, different degrees of homology were found among the 26 ruminococcal clones. The difficulty of subcloning the ruminococcal DNA fragments into plasmid vectors was one of the major obstacles in the study of cellulase gene in foreign hosts. Vectors with different functions were tried but all the recombinant plasmid clones showed instability. The cellular location of cellulase enzymes in E. coli cell was determined. Most of the endo- and exo-glucanases were found in the periplasmic space. Partial purification of these cellulase enzymes from E. coli cells using chromatography was then performed and the characterization of these enzymes was achieved. Using ExoIII deletion, the locations of endoglucanase and exoglucanase genes were determined in λCM903. The internal HindIII-HindIII fragment of 7.3 kb from λCM903 was sequenced. Five ORFs were detected using computer software (UWGCG) analyses. The first ORF which coded for an endoglucanase gene (renA) was 2157 bp long with putative 680 amino acid residue. The SD sequence and promoter sequence were present. The best fitting cellulase gene tested was that of the cenA gene of Ce. fimi. An unusual structure of Pro-Thr-Ser rich region, which had 38 out of 42 a.a. residue of proline, threonine or serine, was found in the N-terminal of the putative peptide. The second ORF which was 1821 bp long coded for an exoglucanase gene (rex). The putative amino acid sequence had 572 a.a. residue, also a SD sequence and a promoter sequence were found. A Pro-Thr-Ser rich region, which was highly conserved with PTS of renA, was found at the C-terminal of the putative peptide. Again, the cellulase gene, cex gene of Ce. fimi showed the best similarity. It is suggested that the gene structure of cellulase in R. flavefaciens strain 186 was similar to cellulase genes in family A as described by Ong et al, 1989. The third ORF was found overlapping with ORF1 using the transcriptional second reading frame. This ORF had a putative SD sequence but lacked a promoter sequence. The coding region of this ORF has the characteristics of a β-glucosidase gene which was 1300 bp long (a putative sequence of 443 amino acid residues). The fourth ORF which used the second reading frame was 1300 bp long with a SD sequence 5' upstream of the ATG codon, overlapped with ORF1 gene. This ORF coded for a protease gene. The fifth ORF located closely to the 3' end of the 7.3 kb fragment used the second reading frame. This ORF had both putative SD sequence and promoter sequence. It was 1080 bp long with putative 341 amino acid residues and showed the structure of a xylanase gene. A short PTS region was also found in this ORF.